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SAM 109.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 109

Supplemental Assay Method for the Titration of Bovine Rhinotracheitis 

Neutralizing Antibody (Constant Virus-Varying Serum Method)

Date: October 16, 2014

Number: SAM 109.05

Supersedes: SAM 109.04, September 9, 2011

Standard Requirement: 9 CFR 113.216

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals:

/s/Geetha B. Srinivas Date: 01Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 03Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 08Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Titration of Bovine Rhinotracheitis Neutralizing Antibody

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the Test Serum, PCS, and NCS

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendices
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Supplemental Assay Method for the Titration of Bovine Rhinotracheitis Neutralizing Antibody

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test of serum for the serum 

neutralization (SN) antibody titer against bovine rhinotracheitis virus (IBR). The assay uses a 

constant amount of virus to test varying dilutions of serum in a cell culture system. The assay 

meets the requirements in title 9, Code of Federal Regulations (9 CFR), part 113.216, to test 

serum samples for potency testing of inactivated IBR vaccines.

Note: For this SAM, the dilution terminology of 1:2, 1:4, etc. specifies 1 part + 1 part 

(liquid), 1 part + 3 parts, etc.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Incubator, 36°± 2°C, 5% ± 1% CO2, high humidity (Model 3158, Forma 

Scientific, Inc.)

2.1.2 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries, Inc.)

2.1.3 Microscope, inverted light (Model CK, Olympus America, Inc.)

2.1.4 Micropipettor, 200-μL, 500-μL, 1000-μL and tips 

2.1.5 Water bath

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 IBR Reference virus, Cooper strain 

2.2.2 Madin-Darby bovine kidney (MDBK) cells

2.2.3 Minimum essential medium (MEM) 

1. 9.61 g MEM with Earles salts without bicarbonate

2. 2.2 g sodium bicarbonate (NaHCO3)
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Supplemental Assay Method for the Titration of Bovine Rhinotracheitis Neutralizing Antibody

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3. Dissolve Steps 1 and 2 with 900 mL deionized water (DI).

4. Add 5 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60°± 2°C until dissolved. Add to Step 3 with constant stirring.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add:

a. 10 mL L-glutamine

b. 50 μg/mL gentamicin sulfate

8. Store at 4°± 2°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS)

3. Store at 4°± 2°C.

2.2.5 Maintenance Medium

1. 980 mL of MEM

2. Aseptically add 20 mL of gamma-irradiated FBS

3. Store at 4°± 2°C.

2.2.6 Positive IBR control serum (PCS) 

2.2.7 Negative IBR control serum (NCS)

2.2.8 Cell culture plates, 96-well

2.2.9 Polystyrene tubes, 12 x 75-mm
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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in antibody titration assays, cell culture maintenance, and in 

the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

3.2.1 On the day of test initiation, set a water bath at 36°± 2°C.

3.2.2 On the day of test initiation, set a water bath at 56°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 Two days prior to test performance, seed 96-well cell culture plates with 

MDBK cells, in Growth Medium, at a cell count (104.7 to 105.2 cells/mL) that will 

produce a confluent monolayer after 48 ± 8 hours of incubation at 36°± 2°C in a 

CO2 incubator. These become the MDBK Plates. Four Test Sera may be tested 

per MDBK Plate.

3.3.2 On day of test initiation:

1. Working IBR Reference. Rapidly thaw a vial of IBR Reference in a 

36°± 2°C water bath. Dilute the virus in MEM to contain 50-300 50% 

tissue culture infective dose (TCID50)/25 μL.

2. Virus Back Titration. Make 3 serial tenfold dilutions of Indicator 

Virus.

a. Place 900 μL of MEM into 3, 12 x 75-mm polystyrene tubes 

labeled 10-1x 10-3.

b. Transfer 100 μL of Working IBR Reference to the 10-1tube; mix 

by vortexing. Discard pipette tip.

c. Using a new pipette tip, transfer 100 μL from the 10-1tube to the 

10-2tube; mix by vortexing.

d. Using a new pipette tip, transfer 100 μL from the 10-2tube to the 

10-3tube; mix by vortexing.
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3.4 Preparation of the Test Serum, PCS, and NCS

3.4.1 On the day of test initiation, heat inactivate all Test Serum samples, PCS, 

and NCS in a 56°± 2°C water bath for 30 ± 5 minutes.

3.4.2 Prepare serial twofold dilutions of the Test Serum samples, PCS, and NCS 

in a 96-well cell culture plate, which becomes the Dilution Plate (Appendix I). A 

sufficient number of dilutions of the PCS should be performed to reach an 

endpoint for the control. Twofold dilutions are made as follows:

1. Add 150 μL MEM to all wells in Rows B through D. Rows E through 

H may be used if additional samples are to be tested.

2. Add 150 μL Test Serum samples, PCS, or NCS to Rows A and B. Mix 

Row B with the multichannel micropipettor (7 ± 2 fills by aspiration and 

expulsion of the 12-channel micropipettor).

3. Using new pipette tips, transfer 150 μL from Row B to Row C. Mix 

Row C with the multichannel micropippetor (7 ± 2 fills).

4. Repeat as in Section 3.4.2(3) for Row D, and discard 150 μL from all 

wells in Row D. Additional dilutions of Test Serum samples may be 

prepared if endpoint titers are desired.

5. Add 150 μL of Working IBR Reference virus to all wells of the 

Dilution Plate; tap plates gently to mix. Incubate for 60 ± 10 minutes at 

36°± 2°C to allow for neutralization of virus. The addition of virus results 

in an additional twofold dilution of Test Serum.

4. Performance of the Test

4.1 On the day of test initiation, decant Growth Medium from the MDBK Plates.

4.2 Inoculate 50 μL/well of each Virus-Serum mixture (Section 3.4.2(5)) into 5 

wells/dilution of a MDBK Plate (Appendix II).

4.3 Inoculate 25 μL of each dilution of Virus Back Titration into 5 wells of a MDBK 

Plate.

4.4 Add 25 μL of MEM to each Virus Back Titration well

4.5 Maintain 5 or more wells on a MDBK Plate as uninoculated cell controls.
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4.6 Incubate MDBK Plates for 60 ± 10 minutes at 36°± 2°C in a CO2 incubator.

4.7 Add 200 μL/well of Maintenance Medium to all wells (do not remove VirusSerum mix). Incubate MDBK Plate for 4 days ± 12 hours postinoculation at 36°± 2°C in 

a CO2 incubator.

4.8 At 4 days postinoculation, examine the wells with an inverted light microscope at 

100X magnification. The CPE of IBR is visible as grape-like clusters of rounded cells in 

the cell monolayer where the cells have been destroyed by the virus.

Record the number of wells/dilution showing any characteristic CPE of IBR for each Test 

Serum and Virus Back Titration.

4.9 Calculate the titer of the Test Serum, PCS, and NCS using the Spearman-Kärber 

method as commonly modified.

Example:

1:2 dilution of Test Serum = 5/5 wells CPE negative

1:4 dilution of Test Serum = 5/5 wells CPE negative

1:8 dilution of Test Serum = 3/5 wells CPE negative

1:16 dilution of Test Serum = 0/5 wells CPE negative

Spearman-Kärber formula: 

Test Serum titer = (X – d/2 + [d • S]) where:

X = log10 of lowest dilution (= 0.3)

d = log10 of dilution factor (= 0.3)

S = sum of proportion of CPE negative

5 5 3 0 13

5 + 5 + 5 + 5 = 5 = 2.6

Test Serum titer = (0.3 – 0.3/2 + [0.3 • 2.6) = 0.93

antilog of 0.93 = 8.5

From the example, the Test Serum has a SN titer of 1:8.5.

4.10 By the same Spearman-Kärber method, calculate the endpoint of the BVDV Back 

Titration. The titer is expressed as log10 tissue culture infective doses 50 (TCID50) 

per 25 μL dose.
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Supplemental Assay Method for the Titration of Bovine Rhinotracheitis Neutralizing Antibody

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Example:

100dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-1dilution of the Working BVDV = 5 of 5 wells CPE Positive

10-2dilution of the Working BVDV = 3 of 5 wells CPE Positive

10-3dilution of the Working BVDV = 0 of 5 wells CPE Positive

Test Serum titer = (X – d/2 + [d • S]) where:

X = log10 of lowest dilution (= 0)

d = log10 of dilution factor (= 1)

S = sum of proportion of CPE positive

5 5 3 0 13

5 + 5 + 5 + 5 = 5 = 2.6

Test Serum titer = (0 – 1/2 + [1 • 2.6) = 2.1

antilog of 2.1 = 125.9

Titer of the Working BVDV is 126 TCID50/25 μL dose in the test.

5. Interpretation of the Test Results

5.1 Validity requirements

5.1.1 No visible contamination or serum toxicity should be observed in more 

than one well/dilution of any dilution of a Test Sera or the FBS.

5.1.2 The SN titer of the PCS should vary by no more than twofold from its 

mean titer as established from a minimum of 10 titrations that have been 

previously performed.

5.1.3 The SN titer of the NCS should be < 1:2.

5.1.4 The Virus Back Titration must have between 50-300 TCID50/25 μL.

5.2 Four out of 5 postvaccinated Test Sera shall have a SN titer ≥ 1:8 for a 

SATISFACTORY result as stated in 9 CFR 113.216.

5.3 Retest

5.3.1 If the initial test is valid and less than 4 out of 5 postvacinnated Test Sera 

have a SN titer < 1:8, the test is repeated (1st retest).
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5.3.2 If the 2nd valid test (1st retest) confirms the initial results, the Test Serial 

is UNSATISFACTORY.

5.3.3 If the 2nd valid test (1st retest) fails to confirm the initial result, the Test 

Sera is tested a 3rd time (2nd retest).

1. If 4 out of 5 postvaccinated Test Sera have a SN titer ≥ 1:8 in the 2nd 

and 3rd valid tests (1st and 2nd retests), the Test Serial is 

SATISFACTORY.

2. If the 3rd valid test (2nd retest) confirms the initial result, the Test 

Serial is UNSATISFACTORY.

6. Report of Test Results

Record the SN titers on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.216, U.S. Government Printing 

Office, Washington, DC.

7.2 Finney, DJ. Statistical Method in Biological Assay. 3rd ed. Charles Griffin and 

Company, London, 1978. 

7.3 Rose NR, H Friedman, JL Fahey, eds. Manual of Clinical Laboratory 

Immunology. Chapter 11: Neutralization Assays. ASM, Washington, DC, 1986.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB.
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Version .03

 The Contact information has been updated.

Version .02

 This document was revised to clarify practices currently in use at the Center for 

Veterinary Biologics and to provide additional detail. 

 The assay was converted from a virus plaque method utilizing Bovine Embryonic 

Kidney cell cultures to a microtiter cytopathic effect assay utilizing Madin Darby 

Kidney cell cultures.
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Appendix I

Transfer Plate

A

1:2*

TS1 TS2 TS3 TS4 TS5 TS6 TS7 TS8 TS9 TS 

10

PSC NSC

B 1:4            

C 1:8            

D 1:16            

E 

1:2*

TS11 TS12 TS13 TS14 TS15 TS16 TS17 TS18 TS19 TS20  

F 1:4            

G 1:8            

H 1:16            

*Final serum dilutions

TS= Test Serum (diluted 1:2 - 1:16)

PCS= Positive control serum (final dilutions 1:2-1:16)

NCS= Negative control serum (final dilutions 1:2-1:16)

Appendix II

Test Plate

A 1:2 TS1 TS1 TS1 TS1 TS1 CC CC TS2 TS2 TS2 TS2 TS2

B 1:4

C 1:8

D1:16

E 1:2 TS3 TS3 TS3 TS3 TS3 CC CC TS4 TS4 TS4 TS4 TS4

F 1:4

G 1:8

H 1:16

TS= Test Serum CC= Cell Control